Although HAART is successful to block active replication of HIV-1 in AIDS patients, it does not eradicate viruses. Presence of latent HIV-1 reservoirs remains a major obstacle to the cure. Recently, it was uncovered that the size of latent HIV-1 reservoirs is much larger than previously estimated. Thus, identification of novel host genes that can be targeted for reverting HIV-1 latency is urgent. Our recent studies of host factors modulating HIV-1 replication identified that FACT (facilitates chromatin transcription) proteins, SUPT16H and SSRP1, restrict HIV-1 replication. Our further studies demonstrated that FACT proteins interfere with TAT-LTR transcriptional activities, and depletion of FACT proteins enhances HIV-1 transcription and facilitates reactivation of latent HIV-1. In this proposal, we wil explore molecular mechanisms how FACT proteins negatively regulate HIV-1 replication. Aim 1: Our genetic data showed that depletion of FACT proteins significantly enhance HIV-1 transcription. It was a surprising result considering that FACT proteins generally facilitate transcription. It is critical to study FACT's function under circumstance of HIV-1 infection, which will provide new knowledge about FACT functions that might be unique for HIV-1. We postulate two possible mechanisms that might lead to FACT suppressive activities: (i). Presence of FACT proteins might affect P- TEFb activity in HIV-1 transcriptional elongation; (ii). FACT proteins might process altered nucleosome exchange activity for HIV-1 transcription. These hypotheses will be thoroughly tested by a series of experiments using multidisciplinary approaches. Aim 2: Our preliminary results indicated that SUPT16H might directly bind with HIV-1 TAT and recruit to LTR promoter. However, a lot of information still lacks for further characterization of these interactions. Thus, we will study the molecular details of FACT interactions with HIV-1 TAT-LTR as well as other key host transcriptional factors (PAF1 and P-TEFb). We will (i) map which domain(s) of SUPT16H mediate its direct interaction with TAT; (ii) determine whether LTR recruitment of SUPT16H depends on TAT or PAF1; (iii) evaluate the impact of FACT proteins on P-TEFb and TAT-LTR interactions. Aim 3: If indeed FACT proteins impose an inhibitory effect on HIV-1 transcription, through which they might play a role in regulating HIV-1 latency. Our earlier results using HIV-1 latently infected J-LAT cells confirmed that depletion of FACT proteins by RNAi spontaneously reverts HIV-1 latency. In this aim, we will further evaluate the effects of FACT proteins on HIV-1 latency in a primary CD4+ T cell model: (i) Role of FACT proteins in suppressing HIV-1 transcription for latency establishment will be determined; (ii) Effects of FACT protein depletion on HIV-1 reactivation from latency will be measured. (iii) Coordination of FACT proteins with other HIV-1 transcriptional suppressors, histone deacetylases (HDACs), will be investigated. Above all, we expect that our in-depth functional studies of FACT proteins will shed light in understanding their roles in HIV-1 replication and provide sufficient scientific foundation to target them for HIV-1 anti-latency therapy.